Enzymes Biochemistry Lecture PDF

BIOCHEMISTRY LECTURE (PRELIM) ENZYMES ⊗ From the Greek work “en” meaning “in” and “zyme” meaning “yeast” ⊗ Biologic catalysts ⊚ Hasten chemical reactions Enzyme Nomenclature ⊗ Not consumed during the reacti...

BIOCHEMISTRY LECTURE (PRELIM) ENZYMES ⊗ From the Greek work “en” meaning “in” and “zyme” meaning “yeast” ⊗ Biologic catalysts ⊚ Hasten chemical reactions Enzyme Nomenclature ⊗ Not consumed during the reactions 1. Substrate + -ase ⊚ Lipid = lipase ⊗ Not undergoing a chemical change after the reactions ⊚ Ester= esterase ⊚ Protein= protease 2. Reaction it catalyzes ⊚ Oxidation = oxidase ⊚ Reduction = reductase ⊚ Hydrolysis = hydrolase ⊚ remove hydrogen atoms = dehydrogenase ⊚ remove carboxyl groups = decarboxylase 3. Enzyme Commission Nomenclature (E.C.) ⊚ E.C. 1.1.1.27 ⊚ 1st digit – class ⊚ 2nd digit – subclass ⊚ 3rd and 4th digit – serial number Classification of Enzymes Enzymes Specificity 1. Oxidoreductases ⊗ Enzymes may recognize and catalyze: Oxidation and reduction ⊚ A single substrate 1. Oxidation – removal of H ion ⊚ A group of similar substrates 2. Reduction – accept H ion ⊚ A particular type of bond E.C. 1.1.1.27 : L-lactate NAD+ Oxidoreductase Lactate Dehydrogenase Example Reaction: A + B → A– + B+ Definition of Terms ⊗ Substrate: Acted upon by the enzyme and specific ⊗ Isoenzyme: Different form but with the same action ⊗ Cofactor: Non-protein molecule of a conjugated enzyme 2. Transferases ⊚ Activator/Metal ions: Inorganic cofactor Transfer of functional groups other than ⊚ Coenzyme: Organic cofactor hydrogen from one substrate to another ⊗ Apoenzyme: Polypeptide portion of a conjugated 1. Transaminases: transfer of amino group enzyme and inactive enzyme 2. Kinases: transfer of phosphate group from ⊗ Holoenzyme: Apoenzyme + Cofactor ATP to ADP + phosphorylated product ⊗ Proenyzme E.C. 2.6.1.1 : L-Aspartate: 2- ⊚ Also known as zymogen Oxaloglutarate Aminotransferase ⊚ Inactive form or precursor of enzyme ⊚ It is then converted, usually by proteolysis, Aspartate Aminotransferase Example Reaction: A–X + B → A + B–X to the active form when it has reached the site of its activity  Hydratases catalyze the addition of H2O to a substrate. 3. Hydrolases is an enzyme that catalyzes a hydrolysis reaction in which the addition of a water molecule to a bond causes the bond to break Central to the process of digestion 1. Carbohydrases: breaking of glycosidic bonds in oligosaccharides and polysaccharides 2. Proteases: breaking of peptide linkages in proteins 3. Lipases: breaking of ester linkages in triglycerides E.C. 3.2.1.1 : 1,4-D-Glucan Glucanohydrolase Alpha-Amylase Example Reaction: A–B + H2O → A–OH + B–H 5. Isomerases is an enzyme that catalyzes the isomerization (rearrangement of atoms) of a substrate in a reaction, converting it into a molecule isomeric with itself Only one product and one reactant (substrate) 4. Lyases Example: Phosphoglycerate mutase is an enzyme that catalyzes the addition of a group to a double bond or the removal of a group to form a double bond in a manner that does not involve hydrolysis or oxidation any member of a class of enzymes that catalyze the addition or removal of the 6. Ligases elements of water (hydrogen, oxygen), is an enzyme that catalyzes the bonding ammonia (nitrogen, hydrogen), or together of two molecules into one with carbon dioxide (carbon, oxygen) at the participation of ATP double bonds 1. Dehydratase: removal of the components of water from a double bond 2. Hydratase: addition of water to a double bond Examples: Carbonic Anhydrase and Citrate Lyase Decarboxylases catalyze the removal of CO2 from a substrate. Deaminases catalyze the removal of NH3 from a substrate. Dehydratases catalyze the removal of H2O from a substrate.  Enzyme Kinetics ⊗ Michaelis-Menten Theory ⊚ E + S → E-S → P + E This led to the proposal that enzyme catalysis is a two-step process that consists of an initial adsorption whereby the substrate combines with the enzyme to form a noncovalent enzyme–substrate (ES) complex, followed by a second step in which the ES complex decomposes into product (P) and free enzyme (E). relationship between the velocity of an enzymatic reaction and substrate concentration Vmax is the maximum velocity or rate at which the enzyme catalyzed a reaction. Vₘₐₓ (Maximum Velocity): This is the fastest rate the reaction can proceed at when the enzyme is fully saturated with substrate. It reflects the enzyme's catalytic power. It happens when all enzyme active sites are saturated with substrate. Since the maximum velocity is described to be directly proportional to enzyme concentration, it can therefore be used to estimate enzyme concentration. Km is the concentration of substrates when the reaction reaches half of Vmax Km indicates the amount of substrate needed for a particular enzymatic reaction. Km is measure of how easily the enzyme can be saturated by the substrate. A high Km means a lot of substrate must be present to saturate the enzyme, meaning the enzyme has low affinity for the substrate. On the other hand, a low Km means only a small amount of substrate is needed to saturate the enzyme, indicating a high affinity for substrate. (A) At low concentration of substrate, there is a steep increase in the rate of reaction with increasing substrate concentration. The catalytic site of the enzyme is empty, waiting for substrate to bind, for much of the time, and the rate at which product can be formed is limited by the concentration of substrate which is available. (B) As the concentration of substrate increases, the enzyme becomes saturated with substrate. As soon as the catalytic site is empty, more substrate is available to bind and undergo reaction. The rate of formation of product now depends on the activity of the enzyme itself, and adding more substrate will not affect the rate of the reaction to any significant effect. MODELS OF ENZYME ACTION Lock and Key Theory The specific action of an enzyme with a single substrate can be explained using a Lock and Key analogy first postulated in 1894 by Emil Similarities Fischer. In this analogy, the lock is the enzyme and Both say only one substrate will work when it meets the key is the substrate. Only the correctly the active site of the enzyme. sized key(substrate) fits into the key hole (active Both require an enzyme and a substrate. site) of the lock(enzyme). Differences Lock and Key states that there is no change needed and that only a certain type will fit. However induced fit says the active site will change to help to substrate fit. The 'lock and key' model explains the high specificity of most enzymes. It states that each enzyme has a unique 'shape' at its active site that compliments the particular substrate it acts upon. The 'induced fit' model explains the broad specificity of some enzymes (i.e. why some enzymes can work on many different substrates provided they are similar in structure). Factors that Influence Enzymatic Reactions Induced Fit Theory 1. Substrate Concentration induced-fit theory, which states that the binding of a ⊚ First-order reaction substrate or some other molecule to an enzyme causes a change in the shape of the enzyme so as ⊙ Are those which proceed at a rate to enhance or inhibit its activity. exactly proportional to the concentration of one reactant ⊙ A P ⊙ Rate of reaction is exactly proportional to the rate of disappearance of A or the appearance of P ⊚ Second-order reaction ⊙ Are those in which the rate is proportional to the product of the concentration of two reactants ⊙ A+B P  ⊙ Rate of reaction is exactly proportional to the rate of disappearance of A or the appearance of P ⊚ Zero-order reaction ⊙ The reactions are zero-order with respect to the reactants ⊙ Rate of reaction depends on the concentration of catalysts or on some factor other than the concentration of the molecular species undergoing reaction 4. Temperature ⊚ 37 degrees Celsius (normal) ⊚ Denaturation at 40-50 degrees Celsius ⊚ assay temperatures: 25, 30, or 37 ºC 2. Enzyme Concentration ⊚ The higher the enzyme level, the faster the reaction will proceed 5. Cofactors ⊚ Nonprotein entities that must bind to particular enzymes before a reaction occurs ⊚ Metallic or nonmetallic / organic or inorganic 3. pH 6. Activators ⊚ pH = 7.0 – 8.0 ⊚ Proper substrate binding ⊚ Changes in pH may denature the enzyme ⊚ Linking substrate to the enzyme or ⊚ Protein in nature coenzyme ⊚ Undergoing oxidation or reduction 7. Inhibitors ⊚ Interfere with enzyme reactions ⊙ Reversible Competitive ⊚ Competitive inhibitors physically bind to the active ⊙ Irreversible site of an enzyme and compete with the substrate for ⊚ molecule that inactivates enzymes by forming a the active site. strong covalent bond to an amino acid side-chain ⊚ Inhibitor remains unchanges but its physical group at the enzyme’s active site presence at the site prevents a normal substrate in ⊚ do not have structures similar to that of the enzyme’s occupying the site. normal substrate ⊚ RESULT: decrease enzyme activity ⊚ enzyme is permanently deactivated Measurement of Enzyme Activity ⊗ Increase in product concentration ⊗ Decrease in substrate concentration ⊗ Decrease or increase in coenzyme concentration ⊙ Reversible Noncompetitive ⊗ An increase in the concentration of the altered ⊚ A noncompetitive enzyme inhibitor is a molecule that enzyme decreases enzyme activity by binding to a site on an enzyme other than the active site. Enzymatic Assays ⊚ The substrate can still occupy the active site, but the presence of the inhibitor causes a change in the ⊗ Coupled-Enzymatic Assay structure of the enzyme suffi cient to prevent the ⊚ Substances other than substrate or catalytic groups at the active site from properly coenzyme are necessary and must be effecting their catalyzing action. present in excess ⊚ NAD+ or NADH ⊗ Fixed-Time (Endpoint) Assay ⊚ Reactants are combined ⊚ Reaction proceeds for a designated time ⊚ Reaction is stopped ⊚ Measurement is made of the amount of reaction has occurred ⊗ Continuous-monitoring (Kinetic) Assay ⊚ Multiple measurements ⊚ Absorbance change ⊙ Increasing in absorbance ⊙ Decreasing in absorbance ⊚ Time intervals Calculation of Enzyme Activity International Unit (IU) ⊚ Amount of enzyme that will catalyze the reaction of 1 micromole of substrate per minute under specified conditions International Unit per Liter (IU/L) ⊚ Enzyme concentration Katal Unit (mole/s) ⊚ Amount of enzyme that will catalyze the reaction of 1 mole of substrate per second under specified conditions ⊚ 1.0 IU = 16.7 nkat